FUNCTIONAL ANALYSIS OF CFTR

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The goal of this study is to understand structure/function relationships in CFTR, the protein whose defect causes cystic fibrosis (CF). To do this, we will study the hydrophobic sector of this complex molecule, because this sector of CFTR is most closely associated with the Cl channel function absent in CF. Two broad questions will be asked. One set of experiments will identify elements important to Cl channel activity by implanting cysteine residues to serve as receptors for cysteine-specific reagents. The Cl channel function of such variants will be evaluated in Xenopus oocytes. Among variants that retain function, we will examine more closely those in which channel activity is irreversibly blocked by hydrophilic cysteine-modifying agents. Analysis of a pool of such candidate mutants should identify the subset of transmembrane segments surrounding the CFTR Cl channel and the specific residues lining the channel pore. To complement these studies, parallel experiments will use mutagenesis to identify residues that may interact as salt bridges in CFTR. The genesis of such work relates to conflicting reports regarding the role of two charged residues (E92 and K95) in the first transmembrane segment of CFTR. We believe the present data are resolved if these two interact with one another in a direct way. To test the hypothesis that an ion pair is functionally relevant in this and other areas, we will increase charge density by replacing the anionic partner with a cationic residue (and vice versa) and examine the phenotype of the variant CFTRs in oocytes. To determine whether the original residues interact with one another, we will then replace them, one at a time, with neutral amino acids. In these latter trials, direct evidence for a salt bridge will arise if function is found only when oppositely charged or neutral residues are present. Together, such experiments should add significantly to our understanding of the membrane domain of CFTR, leading to a better grasp of how the CFTR Cl channel functions and is regulated.